Method of treating autoimmune diseases

ABSTRACT

A method of treating an autoimmune disease (for example, Systemic Lupus Erythematosus) comprises administering to the subject a treatment effective amount of a histone hyperacetylating agent, or a pharmaceutically acceptable salt thereof. Methods of screening compounds useful for the treatment of autoimmune disease are also disclosed.

STATEMENT OF FEDERAL SUPPORT

[0001] This invention was made possible with government support undergrant numbers R01 AR39501 from the National Institute of Health. TheUnited States government has certain rights to this invention.

FIELD OF THE INVENTION

[0002] The present invention relates to methods for the treatment ofautoimmune diseases such as systemic lupus erythematosus.

BACKGROUND OF THE INVENTION

[0003] The hallmark of the aberrant cellular immune response in systemiclupus erythematosus (SLE) is T cell dysfunction (A. K. Dayal and G. M.Kammer, Arthritis Rheum. 39, 23 (1996); D. A. Horwitz, et al., inDubois' Lupus Erythematosus., D. J. Wallace and B. H. Hahn, Eds.(Williams & Wilkins, Baltimore, 1997) chap. 10). An imbalance existsbetween exaggerated helper function and deficient cytotoxic/suppressoractivity that promotes inappropriate B cell overproduction ofimmunoglobulins (Ig). The resulting polyclonal hypergammaglobulinemia iscomprised of natural antibodies and pathogenic autoantibodies, includinganti-native DNA. Formation of complement-fixing immune complexes in situor their deposition on vascular endothelium, such as the renalglomerulus, initiates a chronic inflammatory response that leads toirreparable parenchymal damage, ultimately resulting in end-organfailure (R. P. Kimberly, in Arthritis and Allied Conditions: A Textbookof Rheumatology, W. J. Koopman, Ed. (Williams & Wilkins, Baltimore,1997) chap. 27). Moreover, T cell dysfunctions predispose to recurrent,often life-threatening infections (A. G. Iliopoulos and G. C. Tsokos,Sem. Arthritis Rheum. 25, 318 (1996); C. A. Hunter and S. L. Reiner,Curr. Opin. Immunol. 12, 413 (2000)).

[0004] Two principal defects of T cell function in SLE are augmentedexpression of cell surface receptors and altered production ofcytokines. CD40 ligand (CD154) expression is significantly increased andprolonged on both CD4⁺ helper (Th) and CD8⁺ cytotoxic/suppressor (Tc)subpopulations (M. Koshy, et al., J. Clin. Invest. 98, 826 (1996); A.Desai-Mehta, et al, J.Clin.Invest. 97, 2063 (1996)). This prolongedover-expression may be pathophysiologically significant, for binding ofCD154 on Th cells to CD40 on B cells promotes B cell activation and maydrive the polyclonal hypergammaglobulinemia. Moreover, Th2 cellsover-produce IL-10 whereas Th1 cells under-produce IFN-γ. Heightenedlevels of IL-10 may profoundly modify the cellular immune response by(a) downregulating both IFN-γ and IL-2 production by Th1 cells; (b)inhibiting IL-12 generation and down-regulating expression of IL-12receptors on Th1 cells; (c) up-regulating bcl-2 expression andpreventing apoptosis of activated T cells; and, (d) promoting B cellgrowth, differentiation and autoantibody production. By contrast,deficient IFN-γ may significantly hinder cellular immunity in SLE byboth impairing Tc-dependent cytotoxicity and alteringantigen-presentation (B. S. Handwerger, et al., in Lupus: Molecular andCellular Pathogenesis, G. M. Kammer and G. C. Tsokos, Eds. (HumanaPress, Totowa, N.J., 1999) chap. 21).

[0005] While several treatments for SLE and other autoimmune diseaseshave been developed, none are entirely satisfactory. Hence, thereremains a need for new ways to treat autoimmune diseases such as SLE.

SUMMARY OF THE INVENTION

[0006] A first aspect of the present invention is a method of treatingan autoimmune disease in a subject in need thereof, comprisingadministering to the subject a treatment effective amount of a histonehyperacetylating agent, or a pharmaceutically acceptable salt thereof.

[0007] A second aspect of the present invention is a method of treatingSystemic Lupus Erythematosus in a subject in need thereof, comprisingadministering to that subject a treatment effective amount of a histonehyperacetylating agent, or a pharmaceutically acceptable salt thereof.

[0008] A still further aspect of the present invention is the use of anactive agent as described above for the preparation of a medicament forthe treatment of a disorder as described above.

[0009] Still further aspects of the present invention are methods ofscreening candidate compounds for activity in treating autoimmunediseases such as systemic lupus erythematosus.

[0010] Histone deacetylases (HDACs) are enzymes that deacetylatespecific lysine residues of histone amino-terminal tail domains andcertain non-histone substrates. Current evidence implicates thedeacetylases in transcriptional repression (T. Kouzarides, Curr. Opin.Genet. Dev. 9, 40 (1999); W. D. Cress and E. Seto, J. Cell. Physiol.184, 1 (2000)). Complexed with Sin3 and Mi2 transcriptional co-repressorproteins, HDAC/Sin3 and HDAC/Mi2 associate with other DNA-bindingproteins, such as Ikaros (W. D. Cress and E. Seto, J. Cell. Physiol 184,1 (2000); J. Kim et al., Immunity 10, 345 (1999)). These deacetylasecomplexes appear to limit the accessibility of transcription factors tothe promoter by closely juxtaposing the nucleosome to DNA. Of the eighthuman HDACs discovered (W. D. Cress and E. Seto, J. Cell. Physiol. 184,1 (2000)), to date only HDACs1-3 have been identified in T cells (F.Dangond et al., Biochem. Biophys. Res. Comm. 242, 648 (1998)). During Tcell activation, HDAC/Mi2 complexes are recruited to regions of theheterochromatin by Ikaros and modulate gene expression (J. Kim et al.,Immunity 10, 345 (1999); Koipally, J., et al. EMBO J 18, 3090 (1999)).Trichostatin A, an HDAC inhibitor (M. Yoshida, et al., J. Biol. Chem.265, 17174 (1990); S. Finnin et al., Nature 401, 188 (1999)), blocksdeacetylase activity and shifts the equilibrium toward histoneacetylation. By acetylating histones, chromatin is remodeled, promotingaccess of DNA-binding transcription factors and the transcriptionalmachinery to promoter/enhancer regions (W. D. Cress and E. Seto, J.Cell. Physiol 184, 1 (2000); R. D. Kornberg and Y. Lorch, Curr. Opin.Gen. Dev. 9, 148 (1999)). Acetylation may mediate positive or negativeregulatory events that depend upon the particular gene (Z. W. Sun and M.Hampsey, Genetics 152, 921 (1999)). Thus, promoter regions that areordinarily silenced can then be derepressed whereas those that areexpressed can be repressed. However, the use of histone deacetylaseinhibitors or other histone hyperacetylating agents in the treatment ofautoimmune diseases such as SLE has not heretofore been suggested ordisclosed.

[0011] The present invention is explained in greater detail in thespecification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1A shows the down-regulation of CD154 transcript levels byTSA. Increasing concentrations of TSA (0-1000 ng/ml) progressivelyinhibit expression of CD154 mRNA relative to expression of GAPDH mRNA.

[0013]FIG. 1B shows a graphic depiction of a densitometric scan of thegel in FIG. 1A. This graph depicts the percent change of CD154 mRNAexpression with increasing concentrations of TSA over 24 hr. GAPDH mRNAexpression is stable and unchanged in the presence of TSA.

[0014]FIG. 1C shows CD154 transcript levels in T cells incubated in theabsence or presence of 1000 ng/ml TSA over 18 hr. T cells were thenstimulated with 20 ng/ml PMA+0.5 μM IO for intervals to 24 hr. CD154mRNA expression relative to GAPDH mRNA expression is shown.

[0015]FIG. 1D shows a graphic depiction of the percent change in CD154mRNA expression over time in the absence (filled circles) or presence(open circles) of TSA.

[0016]FIG. 1E shows flow cytometric analysis of CD154 and CD3-εexpression on SLE T cells. T cells were cultured in the absence orpresence of 1000 ng/ml TSA for 18 hr, and subsequently activated with 20ng/ml PMA+0.5 μM IO for 24 hr. The abscissa denotes the number of cellsand ordinate the intensity of cell fluorescence signal. Statisticalanalyses were performed by paired Student's t test or one-way ANOVA.

[0017]FIG. 2A shows the down-regulation of IL-10 levels by TSA.Increasing concentrations of TSA (0-1000 ng/ml) progressively inhibitexpression of IL-10 mRNA relative to expression of GAPDH mRNA.

[0018]FIG. 2B shows a graphic depiction of a densitometric scan of thegel in FIG. 2A. This graph depicts the percent change of IL-10transcript expression with increasing concentrations of TSA over 24 hr.

[0019]FIG. 2C shows IL-10 and GAPDH transcripts from T cells of threeSLE subjects. Transcripts from freshly isolated T cells are shown inlanes 1, 4, and 7. Transcripts from T cells cultured for 18 hr in theabsence or presence of 1000 ng/ml TSA are shown in lanes 2, 5, 8 and 3,6, and 9, respectively.

[0020]FIG. 2D shows a graphic depiction of a densitometric scan of thegel in FIG. 2C. This graph shows the percent change in IL-10 mRNA fromSLE T cells cultured in the absence or presence of 1000 ng/ml TSA.

[0021]FIG. 2E illustrates the inhibition of IL-10 secretion byincreasing concentrations of TSA over 24 hr.

[0022]FIG. 2F depicts the percent change of IL-10 production over time.Statistical analysis was performed by paired Student's t test.

[0023]FIG. 3A shows the up-regulation of IFN-γ transcript by TSA. Tcells were incubated in the absence or presence of 1000 ng/ml TSA over18 hr. T cells were then stimulated with 20 ng/ml PMA+0.5 μM IO forintervals to 24 hr. IFN-γ mRNA expression relative to GAPDH mRNAexpression is shown.

[0024]FIG. 3B shows a graphic depiction of a densitometric scan of thegel in FIG. 3A. This graphs depicts the fold increase of IFN-γ mRNA incells cultured in the absence (filled circles) or presence (opencircles) of 1000 ng/ml TSA during intervals to 24 hr.

[0025]FIG. 3C shows IFN-γ protein levels from T cells cultured in theabsence or presence of 1000 ng/ml TSA for 24 hr, and then stimulatedwith 20 ng/ml PMA+0.5 μM IO for 24 hr. The graph shows the fold increaseof IFN-γ protein secretion. Statistical analyses were performed bypaired Student's t test or one-way ANOVA.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0026] The term “treat” as used herein refers to any type of treatmentthat imparts a benefit to a patient afflicted with a disease, includingimprovement in the condition of the patient (e.g., in one or moresymptoms), delay in the progression of the disease, etc.

[0027] The term “pharmaceutically acceptable” as used herein means thatthe compound or composition is suitable for administration to a subjectto achieve the treatments described herein, without unduly deleteriousside effects in light of the severity of the disease and necessity ofthe treatment.

[0028] Active compounds of the present invention may optionally beadministered in conjunction with other compounds useful in the treatmentof the autoimmune disease such as SLE. The other compounds mayoptionally be administered concurrently. As used herein, the word“concurrently” means sufficiently close in time to produce a combinedeffect (that is, concurrently may be simultaneously, or it may be two ormore events occurring within a short time period before or after eachother).

[0029] As used herein, the administration of two or more compounds“concurrently” or “in combination” means that the two compounds areadministered closely enough in time that the presence of one alters thebiological effects of the other. The two compounds may be administeredsimultaneously or sequentially. Simultaneous administration may becarried out by mixing the compounds prior to administration, or byadministering the compounds at the same point in time but at differentanatomic sites or using different routes of administration.

[0030] Autoimmune diseases with which the present invention is concernedinclude, but are not limited to, Rheumatoid Arthritis, Sjogren'sdisease, Polymyositis, Dermatomyositis, and Systemic LupusErythomatosus. A particularly preferred application of the presentinvention is in the treatment of Systemic Lupus erythomatosus (SLE).

[0031] The present invention is primarily concerned with the treatmentof human subjects, but the invention may also be carried out on animalsubjects, particularly mammalian subjects such as mice, rats, dogs,cats, livestock and horses for veterinary purposes, and for drugscreening and drug development purposes. In addition, the presentinvention may be used to treat animal subjects that are models of anautoimmune disease for drug screening and development purposes. Aparticular example of such a model is the mouse NZB/NZW F1 model of SLE.

[0032] 1. Active Compounds.

[0033] Active compounds used to carry out the present invention are, ingeneral, histone hyperacetylating agents, such as histone deacetylaseinhibitors. Numerous such compounds are known. See, e.g., P. Dulski,Histone Deacetylase as Target for Antiprotozoal Agents, PCT ApplicationWO 97/11366 (Mar. 27, 1997). Examples of such compounds include, but arenot limited to:

[0034] A. Trichostatin A and its analogues such as: Trichostatin A(TSA); and Trichostatin C (Koghe et al. 1998. Biochem. Pharmacol.56:1359-1364) (Trichostatin B has been isolated but not shown to be anHDAC inhibitor).

[0035] B. Peptides, such as: Oxamflati [(2E)-5-[3-[(phenylsufonyl)aminol phenyl1]-pent-2-en-4-ynohydroxamic acid (Kim et al. Oncogene,18:2461-2470 (1999)); Trapoxin A (TPX)—Cyclic Tetrapeptide(cyclo-(L-phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-8-oxo-9,10-epoxy-decanoyl))(Kijima et al., J. Biol. Chem. 268, 22429-22435 (1993)); FR901228,Depsipeptide (Nakajima et al., Ex. Cell Res. 241, 126-133 (1998));FR225497, Cyclic Tetrapeptide (H. Mori et al., PCT Application WO00/08048 (Feb. 17, 2000)); Apicidin, Cyclic Tetrapeptide[cyclo(N—O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)](Darkin-Rattray et al., Proc. Natl. Acad. Sci. USA 93, 13143-13147(1996)); Apicidin 1a, Apicidin Ib, Apicidin Ic, Apicidin IIa, andApicidin IIb (P. Dulski et al., PCT Application WO 97/11366); HC-Toxin,Cyclic Tetrapeptide (Bosch et al., Plant Cell 7, 1941-1950 (1995));WF27082, Cyclic Tetrapeptide (PCT Application WO 98/48825); andchlamydocin (Bosch et al., supra).

[0036] C. Hydroxamic Acid-Based Hybrid Polar Compounds (HPCs), such as:Salicylihydroxamic Acid (SBHA) (Andrews et al., International J.Parasitology 30, 761-768 (2000)); Suberoylanilide Hydroxamic Acid (SAHA)(Richon et al., Proc. Natl. Acad. Sci. USA 95, 3003-3007 (1998));Azelaic Bishydroxamic Acid (ABHA) (Andrews et al., supra);Azelaic-1-Hydroxamate-9-Anilide (AAHA) (Qiu et al., Mol. Biol Cell 11,2069-2083 (2000)); M-Carboxycinnamic Acid Bishydroxamide (CBHA) (Riconet al., supra); 6-(3-Chlorophenylureido)carpoic Hydroxamic Acid(3-Cl-UCHA) (Richon et al., supra); MW2796 (Andrews et al., supra); andMW2996 (Andrews et al., supra). Note that analogs not effective as HDACInhibitors are: Hexamethylene bisacetamide (HBMA) (Richon et al. 1998,PNAS, 95:3003-3007); and Diethylbix(pentamethylene-N,N-dimethylcarboxamide) malonate (EMBA) (Richon etal. 1998, PNAS, 95:3003-3007).

[0037] D. Short Chain Fatty Acid (SCFA) Compounds, such as: SodiumButyrate (Cousens et al., J. Biol. Chem. 254, 1716-1723 (1979));Isovalerate (McBain et al., Biochem. Pharm. 53:1357-1368 (1997));Valerate (McBain et al., supra); 4-Phenylbutyrate (4-PBA) (Lea andTulsyan, Anticancer Research, 15, 879-873 (1995)); Phenylbutyrate (PB)(Wang et al., Cancer Research, 59, 2766-2799 (1999)); Propionate (McBainet al., supra); Butrymide (Lea and Tulsyan, supra); Isobutyramide (Leaand Tulsyan, supra); Phenylacetate (Lea and Tulsyan, supra);3-Bromopropionate (Lea and Tulsyan, supra); and Tributyrin (Guan et al.,Cancer Research, 60, 749-755 (2000)).

[0038] E. Benzamide derivatives, such as: MS-27-275[N-(2-aminophenyl)-4-[N-(pyridin-3-yl-methoxycarbonyl) aminomethyl]benzamide] (Saito et al., Proc. Natl. Acad. Sci. USA 96, 4592-4597(1999)); and 3′-amino derivative of MS-27-275 (Saito et al., supra).

[0039] F. Other inhibitors, such as: Depudecin [its analogues(mono-MTM-depudecin and depudecin-bisether) do not inhibit HDAC) (Kwonet al. 1998. PNAS 95:3356-3361); and Scriptaid (Su et al. 2000 CancerResearch, 60:3137-3142).

[0040] The active compounds disclosed can, as noted above, be preparedin the form of their pharmaceutically acceptable salts. Pharmaceuticallyacceptable salts are salts that retain the desired biological activityof the parent compound and do not impart undesired toxicologicaleffects. Examples of such salts are (a) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; and saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; (b)salts formed from elemental anions such as chlorine, bromine, andiodine, and (c) salts derived from bases, such as ammonium salts, alkalimetal salts such as those of sodium and potassium, alkaline earth metalsalts such as those of calcium and magnesium, and salts with organicbases such as dicyclohexylamine and N-methyl-D-glucamine.

[0041] 2. Compounds for Concurrent Administration.

[0042] The active compound histone hyperacetylating agents describedherein may be administered concurrently with other active compoundsknown for the treatment of autoimmune diseases (such as systemic lupuserythematosus). Examples of such other active compounds include, but arenot limited to: (i) corticosteroids such as prednisolone sodiumphosphate, such as Pediapred®; methylprednisolone, such as Medrol®;prednisone, such as Deltasone® or Orasone®; and dexamethasone, such asDecadron® Tablets; (ii) steroids such as lynestrenol—a progestagen;desogestrel—a progestagen; ethylestrenol—an anabolic steroid; andtibolone—a weak progestational, anabolic, androgenic steroid (H. A.Verheul et al. Clin. Immunol. Immunopathol. 38:198-208 (1986)); andexogenous DHEA—dehydroepiandrosterone—(T. Suzuki et al. Clin. Exp.Immunol. 99:251-255 (1995)); and (iii) other compounds such ashydroxchloroquine sulfate, such as Plaquenil®; H1-A (isolated fromCordyceps sinensis) (L. Y. Yang, et al. J. Lab Clin. Med. 134:492-500(1999)); sulfasalazine (a.k.a. Salazosulfapyridine) (E. Delaporte et al.Ann. Dermatol. Venereol. 124:151-156 (1997)); anti-ICAM-1—murineantiintercellular adhesion molecule-1 (R. L. Brey et al. Lupus 6:645-651(1997)); MX-68—upolyglutamable antifolate (M. Mihara et al. Int. Arch.Allergy Immunol. 13:454-459 (1997)); FK506-(K. Yamamoto et al.Immunology 69:222-227 (1990)); AS101—organotellurium compound—(J.Alcocer-Varela et al. Clin. Exp. Immunol. 77:319-323 (1989));HWA-131-(3-(3,5-ditert.butyl-4-hydroxyphenyl)-7H-thiazolo(3,2-b)(1,2,4)triazin-7-one) (R. R. Bartlett et al. Drugs Exp. Clin. Res. 15:521-526(1989)); and Auranofin—Oral gold compound—(K. Dalziel et al. Br. J.Dermatol. 115:211-216 (1986)).

[0043] The foregoing may be administered in the same formulation and/orby the same route of administration, or by a different formulationand/or different route of administration, as the active agent histonehyperacetylating agents described herein, in their conventional dosagesor dosages which can be determined from the conventional dosages.

[0044] 3. Pharmaceutical Formulations.

[0045] The active compounds described above may be formulated foradministration in a pharmaceutical carrier in accordance with knowntechniques. See, e.g., Remington, The Science And Practice of Pharmacy(9^(th) Ed. 1995). In the manufacture of a pharmaceutical formulationaccording to the invention, the active compound (including thephysiologically acceptable salts thereof) is typically admixed with,inter alia, an acceptable carrier. The carrier must, of course, beacceptable in the sense of being compatible with any other ingredientsin the formulation and must not be deleterious to the patient. Thecarrier may be a solid or a liquid, or both, and is preferablyformulated with the compound as a unit-dose formulation, for example, atablet, which may contain from 0.01 or 0.5% to 95% or 99% by weight ofthe active compound. One or more active compounds may be incorporated inthe formulations of the invention, which may be prepared by any of thewell known techniques of pharmacy consisting essentially of admixing thecomponents, optionally including one or more accessory ingredients.

[0046] The formulations of the invention include those suitable fororal, rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral(e.g., subcutaneous, intramuscular, intradermal, intraperitoneal,intravenous, etc.), topical (e.g., both skin and mucosal surfaces,including airway surfaces) and transdermal administration, although themost suitable route in any given case will depend on the nature andseverity of the condition being treated and on the nature of theparticular active compound which is being used.

[0047] Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above). In general, the formulations of the invention are preparedby uniformly and intimately admixing the active compound with a liquidor finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet may be prepared bycompressing or molding a powder or granules containing the activecompound, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing, in a suitable machine, thecompound in a free-flowing form, such as a powder or granules optionallymixed with a binder, lubricant, inert diluent, and/or surfaceactive/dispersing agent(s). Molded tablets may be made by molding, in asuitable machine, the powdered compound moistened with an inert liquidbinder.

[0048] Formulations suitable for buccal (sub-lingual) administrationinclude lozenges comprising the active compound in a flavoured base,usually sucrose and acacia or tragacanth; and pastilles comprising thecompound in an inert base such as gelatin and glycerin or sucrose andacacia.

[0049] Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The formulations may bepresented in unit\dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising an active compoundas described above, or a salt thereof, in a unit dosage form in a sealedcontainer. The compound or salt is provided in the form of alyophilizate which is capable of being reconstituted with a suitablepharmaceutically acceptable carrier to form a liquid compositionsuitable for injection thereof into a subject. The unit dosage formtypically comprises from about 10 mg to about 10 grams of the compoundor salt. When the compound or salt is substantially water-insoluble, asufficient amount of emulsifying agent which is physiologicallyacceptable may be employed in sufficient quantity to emulsify thecompound or salt in an aqueous carrier. One such useful emulsifyingagent is phosphatidyl choline.

[0050] Formulations suitable for rectal administration are preferablypresented as unit dose suppositories. These may be prepared by admixingthe active compound with one or more conventional solid carriers, forexample, cocoa butter, and then shaping the resulting mixture.

[0051] Formulations suitable for topical application to the skinpreferably take the form of an ointment, cream, lotion, paste, gel,spray, aerosol, or oil. Carriers which may be used include petroleumjelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers,and combinations of two or more thereof.

[0052] Formulations suitable for transdermal administration may bepresented as discrete patches adapted to remain in intimate contact withthe epidermis of the recipient for a prolonged period of time.Formulations suitable for transdermal administration may also bedelivered by iontophoresis (see, for example, Pharmaceutical Research 3(6):318 (1986)) and typically take the form of an optionally bufferedaqueous solution of the active compound. Suitable formulations comprisecitrate or bis\tris buffer (pH 6) or ethanol/water and contain from 0.1to 0.2M active ingredient.

[0053] Further, the present invention provides liposomal formulations ofthe compounds disclosed herein and salts thereof. The technology forforming liposomal suspensions is well known in the art. When thecompound or salt thereof is an aqueous-soluble salt, using conventionalliposome technology, the same may be incorporated into lipid vesicles.In such an instance, due to the water solubility of the compound orsalt, the compound or salt will be substantially entrained within thehydrophilic center or core of the liposomes. The lipid layer employedmay be of any conventional composition and may either containcholesterol or may be cholesterol-free. When the compound or salt ofinterest is water-insoluble, again employing conventional liposomeformation technology, the salt may be substantially entrained within thehydrophobic lipid bilayer which forms the structure of the liposome. Ineither instance, the liposomes which are produced may be reduced insize, as through the use of standard sonication and homogenizationtechniques.

[0054] Of course, the liposomal formulations containing the compoundsdisclosed herein or salts thereof, may be lyophilized to produce alyophilizate which may be reconstituted with a pharmaceuticallyacceptable carrier, such as water, to regenerate a liposomal suspension.

[0055] Other pharmaceutical compositions may be prepared from thewater-insoluble compounds disclosed herein, or salts thereof, such asaqueous base emulsions. In such an instance, the composition willcontain a sufficient amount of pharmaceutically acceptable emulsifyingagent to emulsify the desired amount of the compound or salt thereof.Particularly useful emulsifying agents include phosphatidyl cholines,and lecithin.

[0056] In addition to active compounds or their salts, thepharmaceutical compositions may contain other additives, such aspH-adjusting additives. In particular, useful pH-adjusting agentsinclude acids, such as hydrochloric acid, bases or buffers, such assodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodiumborate, or sodium gluconate. Further, the compositions may containmicrobial preservatives. Useful microbial preservatives includemethylparaben, propylparaben, and benzyl alcohol. The microbialpreservative is typically employed when the formulation is placed in avial designed for multidose use. Of course, as indicated, thepharmaceutical compositions of the present invention may be lyophilizedusing techniques well known in the art.

[0057] 4. Dosage and Routes of Administration.

[0058] As noted above, the present invention provides pharmaceuticalformulations comprising the active compounds (including thepharmaceutically acceptable salts thereof), in pharmaceuticallyacceptable carriers for oral, rectal, topical, buccal, parenteral,intramuscular, intradermal, or intravenous, and transdermaladministration.

[0059] The therapeutically effective dosage of any specific compound,the use of which is in the scope of present invention, will vary fromcompound to compound and patient to patient, and will depend uponfactors such as the age, weight, and condition of the patient and theroute of delivery. As a general proposition, a dosage from about 0.01 or0.1 to about 50, 100 or 500 mg/kg will have therapeutic efficacy, withall weights being calculated based upon the weight of the activecompound, including the cases where a salt is employed. Toxicityconcerns at the higher level may restrict intravenous dosages to a lowerlevel such as up to about 10 mg/kg, with all weights being calculatedbased upon the weight of the active base, including the cases where asalt is employed. A dosage from about 10 mg/kg to about 50 mg/kg may beemployed for oral administration. Typically, a dosage from about 0.5mg/kg to 5 mg/kg may be employed for intramuscular injection. Preferreddosages are 1 μmol/kg to 50 μmol/kg, and more preferably 22 μmol/kg and33 μmol/kg of the compound for intravenous or oral administration. Theduration of the treatment is usually once per day for a period of two tothree weeks or until the condition is essentially controlled. Lowerdoses given less frequently can be used prophylactically to prevent orreduce the incidence of recurrence of the disorder, or the severity ofsymptoms. For example, the trichostatin analog SAHA is being given inphase I clinical trials for cancer by an intraveneous route.

[0060] 5. Screening Assays.

[0061] The present invention also provides screening assays foridentifying compounds useful, or potentially useful, in the treatment ofautoimmune diseases such as SLE. Such assays may be carried out inaccordance with known techniques, such as the formats described in P.Dulski, PCT Application WO97/11366 (Mar. 27, 1997).

[0062] One method of screening compounds for activity in treating anautoimmune disease, comprises

[0063] (a) contacting a histone deacetylase, or an extract containinghistone deacetylase with (i) a known amount of a labeled compound thatinteracts with a histone deacetylase; and (ii) a known dilution of atest compound or natural product extract; and then

[0064] (b) determining the inhibition of interaction of said labeledcompound with said histone deacetylase induced by said test compound,where the inhibition of interaction of said labeled compound with saidhistone eacetylase indicates said compound or extract is a candidate forthe treatment of an autoimmune disease.

[0065] The histone deacetylase is preferably a mammalian (e.g., mouse,rat, rabbit) histone deacetylase, and is most preferably a human histonedeacetylase. The labeled compound may be any of the active agentsdescribed above, labeled with a suitable detectable group such astritium. In general, the labeled compound will be one which binds tohistone deacetylase or is a substrate of histone deacetylase. The testcompound may be of any source, such as an oligomer or a non-oligomerfrom a combinatorial library, or a rationally synthesized candidatecompound. Extracts may be obtained from any suitable source, such asplant extracts obtained through techniques known in traditional, folk orherbal medicine. The determining step may be carried out qualitativelyor quantitatively by any suitable means, such as by Scatchard analysiswith a series of serial dilutions of the test compound or extract.

[0066] In another embodiment, a method of screening compounds foractivity in treating an autoimmune disease such as SLE comprises:

[0067] (a) contacting an intact host cell in vivo or in vitro with atest compound or a natural product extract; and then

[0068] (b) determining the level of histone acetylation in said cell,wherein elevated levels of histone acetylation indicates said compoundor extract is a candidate for the treatment of an autoimmune disease.Where the contacting step is carried out in vivo (e.g., as in the courseof a clinical trial) the compound is administered to a suitable subjectcarrying the cell by any of the same techniques described above foradministering active agents, and the cell (or collection of cells)subsequently collected from the subject for use in the determining step.The cell (or subject) is preferably mammalian (e.g., a mouse, rat orrabbit cell) and in one particularly preferred embodiment is human.Lymphocytes are particularly preferred cells. The subject may be oneafflicted with an autoimmune disease such as SLE (including models ofsuch a disease), or may be a normal (or unafflicted) subject. Elevatedlevels may be determined by comparison to an untreated, control subjector cell, by comparison to levels found in the the same subject or cellor cell population prior to treatment, etc. Assays for histone levelsmay be carried out by any suitable technique, with histone level assaysbeing known to those skilled in the art.

[0069] The examples, which follow, are set forth to illustrate thepresent invention, and are not to be construed as limiting thereof. Inthe following examples, hr means hour; min means minute; TSA meansTrichostatin A; SLE means systemic lupus erythematosus; RT-PCR meansreverse transcriptase polymerase chain reaction; IO means ionomycin, PMAmeans phorbol myristate acetate, ml means milliliter; ng means nanogram;and all temperatures, unless otherwise indicated, are in degreesCelsius.

EXAMPLE 1 Down-Regulation of CD154 Transcript and Protein Levels by TSA

[0070] Because SLE T cells are often activated (D. T. Y. Yu et al., J.Exp. Med. 151, 91 (1980).), the up-regulation of CD154 and IL-10 anddown-regulation of IFN-γ may reflect skewed gene expression due toenhanced recruitment of HDACs to the promoters of these genes. Theresulting dysequilibrium of acetylation might be expected to alter thechromatin structure of the promoters (R. D. Kornberg and Y. Lorch, Curr.Opin. Gen. Dev. 9, 148 (1999)), thereby activating previously silencedgenes while repressing expressed genes. To determine if TSA candown-regulate CD154 transcript expression, T cells from eight SLEsubjects were treated with increasing concentrations of TSA over 18 hr.

[0071] T cells were cultured in the absence or presence of increasingconcentrations of TSA for 18 hr in 5% CO₂ at 37° C. RNA was isolated,cDNAs were prepared, and RT-PCR was performed as previously detailed (D.Laxminarayana, et al., J. Clin. Invest. 92, 2207 (1993)). The primersused were: CD154: 5′-GAATCCTCAAATTGCGGCAC-3′ and5′-CAGAAGGTGACTTGGCATAG-3′; GAPDH: 5′-GGTGAAGGTCGGAGTCAACG-3′ and5′-CAAAGTTGTCATGGATGACC-3′; IL-10: 5′-TTGCCTGGTCCTCCTGACTG-3′ and5′-GATGTCTGGGTCTTGGTTCT-3′; IFN-γ:5′-ATGAAATATACAAGTTATATCTTGGCTTT-3′ and5′-GATGCTCTTCGACCTCGAAACAGCAT-3′.

[0072] The reaction mixtures were subjected to 30 cycles of denaturation(94° C., 1 min) and annealing for 1 min at 53° C. (CD154), 40° C.(GAPDH) and 55° C. (IL-10 and IFN-γ). Extension was for 2 min at 72° C.with a final extension of 7 min at 72° C. using a DNA thermal cycler(Perkin-Elmer).

[0073]FIGS. 1A and 1B demonstrate that TSA maximally inhibits CD154transcript by 60%, but does not modify GAPDH mRNA expression. When SLE Tcells were activated with phorbol myristate acetate (PMA) and ionomycin(IO), CD154 mRNA content increased 100%, peaked at 3 hr, and wanedthereafter (FIGS. 1C and 1D). Under these conditions, however, GAPDHmRNA remained stable, demonstrating that cellular activation also doesnot modify the expression of this gene. By contrast, when T cells werepreincubated with TSA for 18 hr and then activated by PMA+IO overintervals to 24 hr, up-regulation of CD154 transcript was significantlyreduced throughout the entire time course compared to cells not exposedto TSA (FIGS. 1C and 1D; P<0.001). Thus, in SLE T cells TSAsignificantly down-regulates CD154 transcript expression.

[0074] In agreement with previous work (M. Koshy, et al., J. Clin.Invest. 98, 826 (1996); A. Desai-Mehta, et al., J. Clin. Invest. 97,2063 (1996)), we find that an increased proportion of SLE T cellsexpress cell-surface CD154 compared to normal and disease controls. Todetermine if TSA-dependent down-regulation of CD154 mRNA reduces surfaceexpression of CD154, SLE T cells were treated for 18 hr with TSA and theproportion of CD154⁺ cells quantified by flow cytometry (E. Hagiwara, etal., Arthritis Rheum. 39, 379 (1996)). Compared with untreated cells,TSA did not effect any significant reduction of cell-surface CD154⁺cells over 24 hr (FIG. 1E). However, activation of SLE T cells withPMA+IO over 24 hr induced a new population of CD154⁺ cells that wascompletely inhibited when cells were pretreated with TSA prior toactivation (FIG. 1E; P=0.005). By contrast, CD3-ε expression remainedstable under these varying conditions, indicating that TSA's effect onCD154 surface expression is not generalized (FIG. 1E). T cells werestained with saturating concentrations of monoclonal FITC-anti-CD3 andPE anti-CD154 antibodies (Caltag Labs, Burlingame, Calif.) for 30 min at4° C., and the proportion of cells expressing CD3-ε and CD154 wasquantified. In sum, these experiments reveal that TSA down-regulatesboth CD154 mRNA and protein expression, but not GAPDH mRNA or CD3-εexpression, in SLE T cells.

EXAMPLE 2 Down-Regulation of IL-10 Transcript and Protein Levels by TSA

[0075] T cells from SLE subjects produce markedly increased amounts ofIL-10 resulting in high serum levels of the cytokine (B. S. Handwerger,et al., in Lupus: Molecular and Cellular Pathogenesis, G. M. Kammer andG. C. Tsokos, Eds. (Humana Press, Totowa, N.J., 1999), chap. 21; E.Hagiwara, et al., Arthritis Rheum. 39, 379 (1996)). To determine whetherTSA could down-regulate IL-10, a dose-response analysis was performed.Like CD154, increasing concentrations of TSA progressively inhibitedIL-10 transcript expression (FIGS. 2A and 2B). In fact, based onsensitive reverse transcriptase-polymerase chain reaction (RT-PCR)analyses, no detectable IL-10 mRNA was identified at TSA concentrationsof 700-800 ng/ml. By comparison, increasing concentrations of TSA didnot modify GAPDH transcript expression (FIGS. 2A and 2B). As shown inFIG. 2C, IL-10 transcripts were present in freshly isolated T cells (0hr; lanes 1, 4, 7) and remained stable relative to GAPDH transcriptsafter culturing cells for 18 hr (lanes 2, 5, 8). However, when SLE Tcells were cultured in the presence of TSA for 18 hr, no detectableIL-10 transcripts were identified (FIG. 2C, lanes 3, 6, 9). When IL-10transcripts from all eight SLE subjects were quantified relative toGAPDH transcripts, TSA inhibited expression of IL-10 mRNA by 71% (FIG.2D; P=0.029). Treatment of T cells from eight SLE subjects over 18 hrwith increasing concentrations of TSA resulted in a dose-dependentinhibition of IL-10 protein production that was maximal at 300 ng/ml ofthe inhibitor (FIG. 2E). IL-10 and IFN-γ protein production werequantified by ELISA (R & D Systems, Minneapolis, Minn.). Within 6 hr,TSA inhibited IL-10 production by 90%; at 24 hr, there was completeinhibition of IL-10 synthesis (FIG. 2F). Thus, like CD154, TSA was ableto block expression of IL-10 transcript, abolishing IL-10 production bySLE T cells.

EXAMPLE 3 Up-Regulation of IFN-γTranscript and Protein Levels by TSA

[0076] Low production of IFN-γ by SLE T cells may reflectdown-regulation of gene expression (B. S. Handwerger, et al., in Lupus:Molecular and Cellular Pathogenesis, G. M. Kammer and G. C. Tsokos, Eds.(Humana Press, Totowa, N.J., 1999), chap. 21; E. Hagiwara, et al.,Arthritis Rheum. 39, 379 (1996)). To establish whether TSA canup-regulate IFN-γ expression, SLE T cells were treated for 18 hr in theabsence or presence of TSA. During that time, TSA induced a three-foldincrease in IFN-γ transcript compared to untreated cells (FIG. 3A, lanes1 and 7, and FIG. 3B). When T cells were activated with PMA+IO in theabsence of TSA, peak IFN-γ transcript expression increased 13-fold at 1hr over basal levels relative to GAPDH transcript, but waned thereafter.By contrast, activation of T cells in the presence of TSA induced a peak37-fold increase in IFN-γ mRNA at 6 hr over untreated cells relative toGAPDH (FIGS. 3A and 3B; P=0.031). Thus, TSA up-regulated expression ofIFN-γ transcripts in SLE T cells, yielding both a significantlyincreased and prolonged expression of the transcript.

[0077] This strong up-regulation of IFN-γ transcript was reflected insignificantly increased production of IFN-γ protein by 24 hr. In theabsence of stimulation, SLE T cells failed to produce any IFN-γ over 72hr. When T cells were activated with PMA +IO for 24 hr, IFN-γ productionincreased about 24-fold. However, activation of T cells in the presenceof TSA further enhanced IFN-γ output by >12-fold (P=0.011) (FIG. 3C).Taken together, these results demonstrate that TSA rapidly up-regulatesboth IFN-γ transcript and protein production by SLE T cells.

[0078] The capacity of TSA to down-regulate cell surface CD154 and IL-10production and to up-regulate IFN-γ synthesis in SLE T cells providesnew evidence in support of the proposition that skewed gene expressionmay be a fundamental mechanism underlying both the cellular and humoralimmune dysregulation in this disease. That TSA was able to modify thisaltered gene expression in vitro also supports the concept that HDACsmay be recruited to the promoter regions of these genes where theyeffect skewed expression. Because the precise mechanism by which histoneacetylation modifies transcription still remains uncertain (T.Kouzarides, Curr.Opin.Genet.Dev. 9, 40 (1999)), it is also unclear howinhibition of HDAC activity by TSA effects down-regulation of CD154 andIL-10 and up-regulation of IFN-γ in SLE T cells. Notwithstanding, thiscapacity of TSA to modulate the expression of these genes appears tohave the salutary effect of normalizing their protein expression invitro. Because it can simultaneously target multiple genes involved inthe immunopathogenesis of lupus, TSA would be an effective therapeuticagent.

[0079] In SLE, a chronic inflammatory response progressively destroysorgan parenchyma, ultimately leading to irreversible end-organ failuresuch as end-stage renal disease. The immunopathogenesis of this chronicinflammatory process is in part due to the presence of complement-fixingimmune complexes. Formation of pathogenic immune complexes depends onproduction of autoantibodies, such as anti-native DNA, that arise fromdysregulated B cell clones (B. H. Hahn, New Engl. J: Med. 338. 1359(1998)). Therefore, down-regulation of CD154 and IL-10 should eliminateboth the sustained CD154-CD40 interaction as well as high cytokinelevels that drive polyclonal hypergammaglobulinemia and autoantibodyproduction, reducing immune complex formation. Similarly, up-regulationof IFN-γ production might be expected to normalize an abnormal cellularimmune response that predisposes to infections.

[0080] The foregoing is illustrative of the present invention, and isnot to be construed as limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

What is claimed is:
 1. A method of treating an autoimmune disease in asubject in need thereof, comprising administering to said subject atreatment effective amount of a histone hyperactylating agent, or apharmaceutically acceptable salt thereof.
 2. A method according to claim1, wherein said histone hyperacetylating agent is a histone deacetylaseinhibitor.
 3. A method according to claim 1, wherein said histonehyperacetylating agent is selected from the group consisting ofTrichostatin A and Trichostatin C.
 4. A method according to claim 1,wherein said histone hyperacetylating agent is selected from the groupconsisting of Oxamflati, Trapoxin A, FR901228, Apicidin, HC-Toxin,WF27082, and Chlamydocin.
 5. A method according to claim 1, wherein saidhistone hyperacetylating agent is selected from the group consisting ofSalicylihydroxamic Acid, Suberoylanilide Hydroxamic Acid, and AzelaicBishydroxamic Acid.
 6. A method according to claim 1, wherein saidhistone hyperacetylating agent is selected from the group consisting ofAzelaic-1-Hydroxamate-9-Anilide, M-Carboxycinnamic Acid Bishydroxamide,6-(3-Chlorophenylureido)carpoic Hydroxamic Acid, MW2796, and MW2996. 7.A method according to claim 1, wherein said histone hyperacetylatingagent is selected from the group consisting of Sodium Butyrate,Isovalerate, Valerate, 4-Phenylbutyrate, Phenylbutyrate, Propionate,Butrymide, Isobutyramide, Phenylacetate, 3-Bromopropionate, andTributyrin.
 8. A method according to claim 1, wherein said histonehyperacetylating agent is selected from the group consisting ofMS-27-275 and the 3′-amino derivative thereof.
 9. A method according toclaim 1, wherein said histone hyperacetylating agent is selected fromthe group consisting of Depudecin and Scriptaid.
 10. A method accordingto claim 1, wherein said autoimmune disease is selected from the groupconsisting of rheumatoid arthritis, Sjogren's disease, polymyositis, anddermatomyositis.
 11. A method of treating systemic lupus erythematosusin a subject in need thereof, comprising administering to said subject atreatment effective amount of a histone hyperactylating agent, or apharmaceutically acceptable salt thereof.
 12. A method according toclaim 11, wherein said histone hyperacetylating agent is a histonedeacetylase inhibitor.
 13. A method according to claim 11, wherein saidhistone hyperacetylating agent is selected from the group consisting ofTrichostatin A and Trichostatin C.
 14. A method according to claim 11,wherein said histone hyperacetylating agent is selected from the groupconsisting of Oxamflati, Trapoxin A, FR901228, Apicidin, HC-Toxin,WF27082, and Chlamydocin.
 15. A method according to claim 11, whereinsaid histone hyperacetylating agent is selected from the groupconsisting of Salicylihydroxamic Acid, Suberoylanilide Hydroxamic Acid,and Azelaic Bishydroxamic Acid.
 16. A method according to claim 11,wherein said histone hyperacetylating agent is selected from the groupconsisting of Azelaic-1-Hydroxamate-9-Anilide, M-Carboxycinnamic AcidBishydroxamide, 6-(3-Chlorophenylureido)carpoic Hydroxamic Acid, MW2796,and MW2996.
 17. A method according to claim 11, wherein said histonehyperacetylating agent is selected from the group consisting of SodiumButyrate, Isovalerate, Valerate, 4-Phenylbutyrate, Phenylbutyrate,Propionate, Butrymide, Isobutyramide, Phenylacetate, 3-Bromopropionate,and Tributyrin.
 18. A method according to claim 11, wherein said histonehyperacetylating agent is selected from the group consisting ofMS-27-275 and the 3′-amino derivative thereof.
 19. A method according toclaim 11, wherein said histone hyperacetylating agent is selected fromthe group consisting of Depudecin and Scriptaid.
 20. A method ofscreening compounds for activity in treating an autoimmune disease,comprising: (a) contacting a histone deacetylase, or an extractcontaining histone deacetylase with (i) a known amount of a labeledcompound that interacts with a histone deacetylase; and (ii) a knowndilution of a test compound or natural product extract; and (b)determining the inhibition of interaction of said labeled compound withsaid histone deacetylase induced by said test compound, where theinhibition of interaction of said labeled compound with said histonedeacetylase indicates said compound or extract is a candidate for thetreatment of an autoimmune disease.
 21. A method according to claim 20,wherein said histone deacetylase is mammalian histone deacetylase.
 22. Amethod according to claim 20, wherein said histone deacetylase is humanhistone deacetylase.
 23. A method according to claim 20, wherein saidlabeled compound binds to histone deacetylase.
 24. A method according toclaim 20, wherein said labeled compound is a substrate of histonedeacetylase.
 25. A method according to claim 20, wherein said autoimmunedisease is systemic lupus erythematosus.
 26. A method of screeningcompounds for activity in treating an autoimmune disease, comprising:contacting an intact host cell with a test compound or a natural productextract; and then determining the level of histone acetylation in saidcell, wherein elevated levels of histone acetylation indicates saidcompound or extract is a candidate for the treatment of an autoimmunedisease.
 27. A method according to claim 26, wherein said cell is amammalian cell.
 28. A method according to claim 26, wherein said cell isa human cell.
 29. A method according to claim 26, wherein saidautoimmune disease is systemic lupus erythematosus.